Nuclear receptors (NRs) are a class of transcription factors that are activated, or repressed, by natural or pharmaceutical ligands that, one once bound to a NR, induce a receptor conformation that modulates the interaction with transcriptional cofactors and/or gene promoters (McKenna and O'Malley, 2002). Among NRs, Rev-Erb alpha (also named NR1D1; nuclear receptors subfamily 1, group D, member 1) regulates the transcription of a large number of genes via recruitment of cofactors and ligands to promoter sequences within the chromatin (Harding and Lazar, 1995; Raghuram S et al., 2007; Yin L et al., 2007). Heme is a physiological ligand of Rev-Erb alpha, having a Kd of 2-3 μM, and inducing a conformational change in Rev-Erb alpha which results in the suppression of the expression of specific target genes (Moore J T et al., 2006).
Rev-Erb alpha is widely expressed, but expression levels are higher in liver, adipose tissue, skeletal muscle, and brain. Rev-Erb alpha is part of the core clock machinery located in mammals in the suprachiasmatic nucleus (SCN) of the hypothalamus that influences peripheral clocks in synergy with other cues including nutrient status (Green C B et al., 2008). The circadian cycle is regulated by several auto-regulatory feedback loops in gene expression. Per2, Arnt and Single-minded (PAS) domain basic helix-loop-helix transcription factor circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1 (BMAL1) modulate Rev-Erb alpha expression which, in turn, modulates BMAL1 and CLOCK transcription by binding response elements in the BMAL1 promoter, leading to the circadian pattern of BMAL1 expression (Sato T K et al., 2004; Kojetin D et al., 2011). Mice deficient in Rev-Erb alpha expression display loss of the diurnal pattern of BMAL1 expression and exhibit alterations in their circadian behavior patterns (Preitner N et al., 2002).
Rev-Erb alpha is also reported to repress the transcription of genes such as Elovl3 (a very long-chain fatty acid elongase; Anzulovich A et al., 2006) and PAI-1 (Plasminogen activator inhibitor 1, a regulator of the fibrinolytic system and modulator of inflammation, atherothrombosis and atherosclerosis; Raspe E et al., 2001). Other reported Rev-Erb alpha target genes are involved in fatty acid/lipid absorption such as Cd36, and Fabp-3 and -4 (Ramakrishnan S et al., 2005) and in inflammatory bone disorders such as osteoarthritis (Chaturvedi P et al., 2006).
Rev-Erb alpha expression is also expressed in vascular smooth and skeletal muscle cells, suggesting that it can modulate inflammation by regulating IkappaBalpha/NFkappaB dependent gene expression (Ramakrishnan S et al., 2005; Migita H et al., 2004). In human macrophages, Rev-Erb alpha expression diminishes the production of cytokines in response to lipopolysaccharide. These data demonstrate the anti-inflammatory role of Rev-Erb alpha (Barish G D et al., 2005; Fontaine C et al., 2008). Recently, it has been reported an important role of Rev-Erb alpha in inflammatory response (WO 2011/022619) and in the hepatic gluconeogenesis (Grant D et al., 2010).
Rev-Erb alpha is also highly induced during adipogenesis (Chawla A and Lazar M, 1993), possibly due to the interaction with heme (Kumar N et al., 2010), and displays biphasic expression profiles during fat cell development both in vivo and in 3T3-L1 preadipocytes, suggesting its involvement in adipocyte differentiation (WO 2003/060106; Fontaine C et al., 2003). Overexpression of Rev-Erb alpha in these cells increases expression of adipogenesis markers, including aP2, PPARgamma and C/EBPalpha, and a small increase in lipid accumulation. Rev-Erb alpha overexpression synergizes with the PPARgamma ligand Rosiglitazone to increase these markers of adipogenesis. In fact, organs with high metabolic activity, including liver and adipose tissue, display circadian rhythm in the expression of genes involved in key metabolic pathways (Ando H et al., 2005). Mice deficient in Rev-Erb alpha display elevated very low-density lipoprotein triglyceride levels, which correlates with elevated serum and liver levels of ApoCIII, a key player in serum triglyceride metabolism (Raspe E et al., 2002).
People with altered sleep-wake pattern and chronic desynchronization, for example night shift workers, have much higher propensity for cardiovascular diseases and metabolic disorders (Suwazono Y et al., 2008; Lund J et al., 2001). In fact, circadian misalignment leads to decreased leptin throughout the entire cycle, increased glucose, despite increased insulin, suggesting decreased insulin sensitivity and increased blood pressure. Rev-Erb alpha has been proposed as a core clock component that coordinates the circadian metabolic response, suggesting a great potential for Rev-Erb alpha ligands for the medical management of diseases that are associated by circadian rhythm-related disorders (Duez H et al., 2009).
Compounds that modulate Rev-Erb alpha activity thus have the potential to contribute to or even to control the crosstalk between circadian and many other physiological processes, as listed above and in particular for lipid homeostasis (Solt L et al., 2011). In fact, Rev-Erb alpha deficient mice display a dyslipidemic phenotype with elevated very low-density lipoprotein triglyceride levels along with increased liver and serum ApoCIII expression (Raspe E et al., 2001; Raspe E et al., 2002).
The first two synthetic, structurally similar Rev-Erb alpha ligands have been identified: recently. The antagonist SR8278 (Kojetin D et al., 2010) and the agonist GSK4112 (Grant D et al., 2010). GSK4112 was identified in a FRET assay as able to dose-dependently increasing the interaction of a peptide derived from NCoR (Nuclear receptor Co-Repressor) with Rev-Erb alpha (Meng Q J et al., 2008). The treatment with GSK4112 decreases Bmal1 expression in cell culture in a dose-dependent manner and induces adipogenesis in 3T3-L1 cells as demonstrated by lipid accumulation and increased expression of key adipogenic genes (Kumar N et al., 2010; Kojetin D et al., 2011). GSK4112 is therefore a Rev-Erb alpha agonist, regulating the expression of Rev-Erb alpha responsive target genes in a manner similar to Rev-Erb alpha physiological ligand, heme (Raghuram S et al., 2007).
Even if no human diseases or disorders have been exclusively attributed to Rev-Erb alpha dysfunctions, more and more studies associate Rev-Erb alpha with the pathological conditions, in particular those associated to the CNS activity but also to lipid homeostasis and metabolism. Very recently, the role of Rev-Erb beta has been highlightended in mice carrying the deletion of the two isoforms. Indeed since the phenotypic characterization of the Rev-Erba deficient mice, the Rev-Erb beta dependant compensation mechanism was raised. As a consequence of the total absence of Rev-Erbs, the authors reported that the circadian rhythms of Rev-Erb alpha and Rev-Er beta deficient mice were severely perturbed. In addition to the major circadian “arrhythmias”, metabolic perturbations have also been measured. Lipid homeostasis and glucose levels were impaired in Rev-Erbs deficient mice.
GSK4112 and SR8278 have been described as Rev-Erb ligand but displayed poor pharmacokinetic properties because of high clearance and rapid metabolism that decrease their bioavailability, limiting their use in in vitro and biochemical studies. Since SR9009 and SR9011 compounds, which are GSK4122 derivatives, have been tested in different mouse models. As published by Solt et al, those two compounds were suitable for in vivo studies. Solt et al., reported that those two compounds, in addition to have an influence on the circadian clock, were also able to improve the metabolic parameters of diet induced obese mice. The changes measured on biochemical parameters were also associated to a modified gene expression profile in metabolic tissues such as liver, skeletal muscle and adipose tissue.
Thus, those recent results have provided important clues about the nodal role of both Rev-Erbs in the control of the circadian clock and the energetic metabolism. The possibility to pharmacologically modulate the activity of Rev-Erbs represent an interesting option to take pathologies related to metabolic disorders, such as T2 Diabetes but also other circadian associated disorders. (Kumar N et al., 2010; Burns T P, 2008; Solt L et al., 2011). Rev-Erb function is essential for a proper control of pathogen induced inflammation. Loss-of function for Rev-Erb may result in exaggerated host mortality upon exposure to pathogenic organisms, such as certain bacteria, viruses or parasites. Treatment with Rev-Erb ligands may prevent the increased mortality and/or morbidity in patients that suffer from infections with life-threatening pathogens.
Methods for identifying synthetic or natural Rev-Erb alpha and/or dual alpha/beta Rev-Erb modulators in association to specific biological mechanisms and/or disorders have been described in the literature (WO 99/67637; WO 2003/060106; WO 2004/053124; WO 2005/076004). However, none of them allowed identifying 6-substituted [1,2,4]triazolo[4,3-b]pyridazines as a chemical scaffold of interest for generating Rev-Erb agonists. In fact, compounds of this chemotype are described in the literature for having distinct properties such as ion channel modulators (US 2011/021521), GABAA receptor agonists (WO 99/67245; US 2009/143385), Benzodiazepine receptors modulators (Guan L P et al., 2010), Kinase modulators (WO 2004/058769; WO 2008/051805) or biocides (JP 54128595; DE 3222342).